JP2009249335A - Remedy of cerebrovascular dementia, and food and drink and auxiliary food containing the remedy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a remedy of cerebrovascular dementia, safely usable over a long period, and to provide a food and drink and an auxiliary food containing the remedy. <P>SOLUTION: Histidine and/or a compound having the histidine at the N terminus exhibits activity for inhibiting neurocyte death by zinc, deeply concerning with the cerebrovascular dementia. The histidine has conventionally been utilized as a supplement, and is a compound of which the safety is highly evaluated. A product useful for the prevention or amelioration of the cerebrovascular dementia safely usable over a long period can be provided by the utilization of the histidine and/or the compound having the histidine at the N terminus. The histidine and/or the compound having the histidine at the N terminus can be either of a synthetic or a natural product. These are not necessarily required to be isolated, and the effects are exhibited only when an effective amount thereof is contained. Therefore, for example, a broth of Etrumeus teres can be used. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a therapeutic agent for cerebrovascular dementia that can prevent or ameliorate cerebrovascular dementia by reducing the neurotoxicity of zinc, and to foods and drinks and supplements containing the therapeutic agent.

  With the advent of a super-aging society, the number of patients with senile dementia is accelerating. It is said that there are various causes for such senile dementia, and in particular, “cerebral vascular dementia” caused by cerebral infarction and cerebral hemorrhage is regarded as a major cause of senile dementia. Yes. Cerebral vascular dementia is caused by abnormalities in the blood flow of the brain due to vascular disorders such as cerebral ischemia and cerebral infarction, resulting in cell death in the surrounding nerve cells, eventually memory loss, learning disorders, etc. This is a disease that causes dementia symptoms (dementia).

  In preventing or treating cerebrovascular recognition, it is effective to suppress neuronal cell death after cerebral ischemia. Currently, administration of a free radical scavenger such as edaravone immediately after a cerebral ischemic attack, and hypothermia after the cerebral ischemic attack are effective for prevention and treatment. Non-Patent Documents 1 to 6 described regarding suppression of this neuronal cell death are described in detail below.

  Non-Patent Documents 1 and 2 describe that zinc plays an important role in the neuronal cell death mechanism after cerebral ischemia. Zinc is an essential element for the human body and is detected throughout the body, but it is particularly abundant in the brain. In the brain, it is found in high concentrations in the hippocampus and cerebral cortex, which are easily affected by cerebral ischemia, and exists as zinc ions in synaptic vesicles. In Non-Patent Documents 1 and 2, it is clarified that zinc ions cause neuronal cell death in cultured neurons by releasing zinc ions into the synaptic cleft together with glutamic acid during neuronal excitation. Furthermore, it has also been shown that abnormal accumulation of zinc occurs in the hippocampal neurons after cerebral ischemia in parallel with neuronal cell death.

  Non-Patent Document 3 discloses that post-ischemia by pre-administration of CaEDTA (Ethylenediamine-N, N, N, N-tetraacetic acid, calcium), a specific chelator of zinc, to experimental animals before ischemia. It is described that neuronal cell death is suppressed and worsening of cerebral infarction is suppressed. From these research results, it is generally recognized that zinc is involved in neuronal cell death after cerebral ischemia.

  Non-Patent Document 4 describes that pyruvate and oxaloacetate suppress the death of cultured neurons by zinc. Furthermore, Non-Patent Document 5 describes that neuronal cell death after cerebral ischemia can be suppressed by administering pyruvic acid in the brain. Non-Patent Document 6 reports that pyruvate and α-ketoglutarate suppress cerebral nerve cell death due to a hypoglycemic state. These studies suggest that organic acids such as pyruvate have the effect of suppressing cranial nerve cell death.

  Patent Document 1 describes that the present inventors have found that carnosine, which is a physiological substance in the human brain, also has an effect of suppressing neuronal cell death due to zinc. In the living body, carnosine is present in a large amount in muscles, and is considered to be contained in a large amount in, for example, chicken and fish lean. Furthermore, the present inventors have found that citric acid or isocitrate has an effect of suppressing neuronal cell death caused by zinc. These acids are a kind of organic acids present in large quantities in fruits and the like.

Koh, JY, 5 others, “The role of zinc in selective neuronal death after transient global cerebral ischemia.”, “Science” "May 1996, Vol. 272, P1013-1016" Lee, JM and 5 others, “Zinc translocation accelerates infarction after mild transient focal ischemia.”, “Neuroscience, 2002 1 Moon, Volume 115, P871-878 Calderone A, 6 others, “Late calcium EDTA rescues hippocampal CA1 neurons from global ischemia-induced death.” , "Journal of Neuroscience (J. Neurosci.)", November 2004, Volume 24, P9903-9913. “Christian, T” and 3 others, “Zinc Independent Cortical Neuronal Death: Contribution of Energy, Falier, Attributable to Loss of NA Plus and Inhibition of Glycorises”, “Journal of Neuroscience (J. Neurosci.) ", May 2000, Volume 20, P3139-3146. Lee, JM and two others, "Protection by Pyruvate against Transient Forebrain Ischemia in Rats.", Journal of Neuroscience (J. Neurosci.) October 2001, Volume 21, RC171, P1-6 Suh, SW, 4 others, “Pyruvate Administered After Severe Hypoglycemia Reduced Neuronal Death and Cognitive Impairment”, “Diabetes”, 200 May, 54, P1452-1458 JP 2007-314467 A

  Finding a component or food or drink that suppresses neuronal cell death caused by zinc leads to identification of a component or food or drink that leads to prevention or improvement of cerebrovascular dementia. So far, as described above, CaEDTA, pyruvate, oxaloacetate, citric acid, isocitrate, and carnosine have been discovered as substances that suppress zinc-induced neuronal cell death.

  CaEDTA exhibits toxicity in vivo due to its action of chelating various metals. For this reason, it is poor in practicality as a drug for preventing or improving dementia. Pyruvate, oxaloacetate, citric acid, and isocitric acid are compounds that are also present in the living body and can be said to be highly safe. However, the therapeutic agent for cerebrovascular dementia containing them as a major component and the food and drink containing the therapeutic agent Things do not exist. Carnosine is expected to be a therapeutic agent for cerebrovascular dementia and food and drink containing the therapeutic agent because it is present in large amounts in chicken or fish meat, but such a product does not exist. Moreover, the free radical removal agent currently used as a therapeutic agent at the time of ischemia needs administration immediately after an ischemic attack, and has no preventive effect. Therefore, in a society where the number of elderly people with dementia increases dramatically, there are safe drugs, food and drink, and functional foods that can prevent neuronal cell death after ischemia for a long time for prevention and treatment. Although necessary, it has not been developed so far and is highly desired.

  Therefore, an object of the present invention is to provide a therapeutic agent for cerebrovascular dementia that can be safely used for a long period of time, and a food and drink and a supplement containing the therapeutic agent.

  As a result of screening for substances that suppress neuronal cell death caused by zinc using the GT1-7 cells derived from the hypothalamus of the mouse, the present inventors have some neuronal cell death inhibitory activities in water extracts of some seafood. I found out. And the component was isolated and identified, and it discovered that an active ingredient was histidine. The present inventors have arrived at the present invention on the basis of a new finding that histidine and a compound having histidine at the N-terminus are effective in suppressing neuronal cell death by zinc.

That is, the therapeutic agent for cerebrovascular dementia of the present invention comprises histidine and / or a compound having histidine at the N-terminus as an active ingredient.
Here, "therapeutic agent for cerebrovascular dementia" is one of the effect of preventing the occurrence of cerebrovascular dementia by taking, the effect of improving the symptoms of cerebrovascular dementia, or It means a substance that has both effects. The histidine and the compound having histidine at the N-terminus may be not only a simple substance but also a salt. In addition, histidine (including histidine in a compound having histidine at the N-terminus) may be any of D-form, L-form, and DL-form, and in particular, D-histidine (having histidine at the N-terminus) (Including histidine in the compound) is less susceptible to enzymatic degradation than L-histidine, which is an L form, and is preferable because it can maintain a concentration necessary for expressing sufficient activity for a long time in vivo. is there.

  In the therapeutic agent for cerebrovascular dementia of the present invention, it is desirable that histidine and the compound having histidine at the N-terminus are derived from seafood. Fish and shellfish contain a large amount of histidine and a compound having histidine at the N-terminus, are easily taken orally, and can promote prevention and improvement.

In addition, foods and drinks or supplements containing therapeutic agents for cerebrovascular dementia can be eaten as normal meals or supplements, so when continuous intake of therapeutic agents for cerebrovascular dementia is required It can be particularly preferably used.
Here, supplementary foods are foods that indicate that a specific health purpose can be expected, including health functional ingredients that affect the physiological functions of the body, etc., and efficacy and safety for each individual product. Indicates foods for specified health use that are reviewed by the government, and special-purpose foods such as foods for the sick.

  The therapeutic agent for cerebrovascular dementia of the present invention, the food and drink for cerebrovascular dementia containing the therapeutic agent, and the supplementary food include histidine and / or a compound having histidine at the N-terminus as an active ingredient. Can be used safely on a daily basis for the prevention or amelioration of cerebrovascular dementia.

  A therapeutic agent for cerebrovascular dementia according to an embodiment of the present invention (hereinafter also simply referred to as “therapeutic agent”), food and drink containing the therapeutic agent (hereinafter also referred to as “therapeutic agent-containing food and drink”), Alternatively, histidine and compounds having N-terminal histidine used in supplements (hereinafter also referred to as “therapeutic supplement supplements”) are purified from natural products as starting materials, and fermentative methods using natural products Any of those obtained in (1) or chemically synthesized may be used. It is not limited by the starting material or the manufacturing process. Histidine and the compound having histidine at the N-terminus may be in the form of a salt. In addition, histidine and a compound having histidine at the N-terminus do not necessarily need to be isolated, and if they are contained in an effective amount, they can prevent or improve cerebrovascular dementia, such as fish broth Can be used. However, the present invention is not limited to this example. As long as an effective amount of histidine and / or a compound having histidine at the N-terminus is contained, various other seafood can be used, and further, it is limited to seafood. It is not something.

  The therapeutic agent may be in any dosage form as long as it is used for normal oral administration or parenteral administration, or may be ingested in the form of food. Examples of dosage forms used for oral administration or parenteral administration include powders, granules, tablets, capsules, intestinal solvents, troches, liquids for internal use, liquids for external use, suspensions, emulsions, syrups, and injections. Infusion, tube feeding liquid, nasal drops, eye drops, ear drops, suppositories, shipping agents, inhalants or ointments can be arbitrarily selected according to the purpose of use. For solid preparations, excipients, binders, disintegrants, lubricants, flavoring agents, stabilizers, and other adjuvants may be used as the active ingredient depending on the purpose, and sugar coating and film coating may be applied from the viewpoint of moisture and taste protection. Also good. Of course, histidine, compounds having an N-terminal histidine, and salts thereof can be freely selected to be suitable for the formulation of the above dosage form.

A therapeutic agent for cerebrovascular dementia, a food / beverage containing therapeutic agent or a supplement containing a therapeutic agent contains histidine and / or a compound having histidine at the N-terminus, or salts thereof, in addition to proteins, sugars, fats Trace elements, vitamins, emulsifiers, fragrances, or acidulants may be blended. The therapeutic agent-containing food and drink and the therapeutic agent-containing supplementary food may be processed forms such as natural liquid foods, tube liquid foods, semi-digested nutritional foods, component nutritional foods, and drinks. In addition, solid or liquid foods or luxury products such as bread, noodles, rice, confectionery (biscuits, cookies, cakes, candy, chocolate, chewing gum, Japanese confectionery, etc.), mass-produced foods such as tofu and processed products thereof, Fermented food such as sake, medicinal liquor, mirin, vinegar, soy sauce, miso, dressing, yogurt, ham, bacon, sausage, mayonnaise, jam, spread, etc. Beverages, soft drinks, sports drinks, alcoholic drinks, fruit wines, sweet fruit wines, teas and other beverages can also be used. In addition, the therapeutic agent, therapeutic agent-containing food or drink, or therapeutic agent-containing supplement is packaged in a package for packaging a therapeutic agent for cerebrovascular dementia, a therapeutic agent-containing food or beverage, or a therapeutic agent-containing supplement. Since the user can widely recognize the function of food and drink by attaching a display to the effect of preventing / improving, the prevention and improvement of dementia can be promoted. The package can be a bag of vinyl or paper, wrapping paper, a box or a package with a cap.
The method for producing a therapeutic agent for cerebrovascular dementia, a therapeutic agent-containing food or drink, or a therapeutic agent-containing supplement according to the present embodiment varies depending on the purpose and dosage form, but is conventionally used in various fields. It can be manufactured and processed by the method.

  For prevention or improvement of cerebrovascular dementia, the dose of histidine and / or a compound having histidine at the N-terminus varies depending on the age, weight, or symptom of the recipient, the route of administration, the dosage form, and the like. Histidine is an essential amino acid, and it is desired to take a sufficient amount from food during the growth period. As nutritional supplements, products containing histidine are also sold, and it is described that approximately 0.5 to 1 g per serving is taken approximately three times a day. From this it is clear that the use of histidine in the amount necessary to achieve the desired effect and the daily intake of histidine resulting from what is present in food do not pose a health hazard. In addition, published papers (eg, Eur. J. Pharm. 2007 February, 557, p236-244) have shown that ingested histidine reaches the brain via blood. ing. There is no major limitation on the intake or dose of histidine, but for the prevention or improvement of cerebrovascular dementia, it is usually 100 mg to 10 g per day, preferably 300 mg to 5 g per day for adults. It is good to do.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.
Example 1
In Example 1, the activity of suppressing neuronal cell death of zinc was examined mainly using various fish and shellfish water extracts. The seafood used was taken near Miyazaki Prefecture. After separating the edible part and the non-edible part of seafood, an organic solvent such as ethanol or water was added, and the mixture was crushed with a blender, etc., and the centrifugal supernatant was collected and concentrated and solidified by lyophilization. The fish broth was desalted and then concentrated and solidified by lyophilization. After solidifying those samples, 1 mL of water was added to 100 mg and mixed well, and then a centrifugal supernatant was obtained. What processed this with the filter of the 0.45 micrometer hole was made into the sample.

Immortalized hypothalamic neurons (GT1-7 cells) are cultured by a conventional method, dispersed with trypsin enzyme, and then added to a serum-free medium (DMEM / F-12) to a concentration of 5 × 10 ⁵ cells / mL. Thereafter, 200 μL each of which was dispensed into a 96-well culture plate (Nunc) was cultured in a carbon dioxide incubator (37 ° C., 7% CO ₂ ) for 24 hours.

To the GT1-7 cells, 2 μL of the prepared seafood extract was administered, and then the aqueous zinc solution was administered to a concentration of 30 μM. Cell viability was measured by WST-1 method 24 hours after zinc administration (n = 6). As a reagent, Cell Counting Kit (manufactured by Dojindo) was used. The WST-1 method is a simple and accurate measurement method for measuring the number of viable cells by measuring intracellular mitochondrial enzyme activity.
In each experiment, the absorbance by the WST-1 method was measured for the control (viable cells when zinc and the test sample were not added) and the viable cells when only zinc was added. As a result, the absorbance of the WST-1 method showing the inhibitory activity of the test sample can be accurately determined even if the experiment date is different.

  Compared with cells administered with only an aqueous zinc solution (30 μM) (cont in FIG. 1), cells exhibiting higher absorbance were evaluated as having neuronal cell death inhibitory activity. As a result, as shown in FIG. 1, it was found that the water extracts of various seafood have high inhibitory activity.

  Table 1 shows the fish and shellfish used in the test, the site of use, the genus, the date of collection, the place, and the method of fishery.

(Example 2)
About the sample which showed strong inhibitory activity, the mode of the cell death was confirmed with the microscope. In addition, the sample was prepared again, the inhibitory activity test was performed several times, and finally the samples (numbers: 12W, 13W, 79W, 80W, 88W, 89W, 98W, 101W) indicated by black bars in FIG. , IWA). In Example 2, the amount of carnosine in these samples was measured. According to Patent Document 1, it is clear that carnosine suppresses cell death caused by zinc, and it is a well-known fact that carnosine is contained to some extent in muscles of seafood. First, in order to verify whether the inhibitory activity obtained in Example 1 was due to carnosine contained in these samples, the amount of carnosine was measured with an HPLC apparatus.

  The HPLC for measuring carnosine (Shimadzu Corporation, Gradient System) uses a device equipped with a porous graphic carbon column (THERMO Fisher Scientific Inc, φ4.6 × 100 mm), and 0.1 to 0.5% trifluoro An acetonitrile solution containing acetic acid (6-10%) was used as the eluent. The number of mM carnosine contained in the 100 mg / mL sample shown in Table 2 was measured.

The cell viability in Table 2 represents the viability when a sample and 30 μM zinc were added. The cell viability when only zinc was added was 10%. The ND in the carnosine (mM) column indicates no measurement.
Although the carnosine concentration was 10 mM or more for cuttlefish, sea bream, and urchin eagle, the dose to the cell culture medium was 5% (10 μL versus 200 μL), so the final carnosine concentration in these samples was 0.6 mM at maximum. It became clear that. According to Patent Document 1, since a minimum of 2 mM of carnosine is required to suppress neuronal cell death due to zinc, the content of carnosine present in these samples may be insufficient to cause inhibitory activity. It was revealed.
From this experiment, it was concluded that the inhibitory activity of these samples was due to components other than carnosine.

(Example 3)
In Example 3, the active ingredients in these samples were isolated. The ingredients were isolated using abundant sardine broth (IWA) with abundant samples and high inhibitory activity. A freeze-dried product of Urume sardine broth was dissolved in water at 100 mg / mL, and the centrifugal supernatant was used as a starting material for purification.

  The active ingredient was purified by HPLC (Shimadzu Corporation, Gradient System). Detection was carried out by measuring ultraviolet absorption at 215 nm, the flow rate was in principle 1 mL / min, and 5 mL / min when using an ODS preparative column. As the column, a preparative ODS column (Shiseido Capsule Pack UG-120 C18, φ25 × 250 mm), Tosoh TSK-GEL Amido-80, φ4.6 × 250 mm were used. In HPLC using an ODS column, an acetonitrile solution containing 0.1% trifluoroacetic acid was eluted with a gradient condition increasing from 0% to 80% acetonitrile in 0 to 30 minutes. In TSK-GEL Amido-80, an acetonitrile solution containing 0.1% trifluoroacetic acid was eluted under a gradient condition that decreased from 100% acetonitrile to 50% in 0 to 30 minutes.

The separated fraction was examined for inhibitory activity by the following method. Immortalized hypothalamic neurons (GT1-7 cells) are cultured by a conventional method, dispersed with trypsin enzyme, and then added to a serum-free medium (DMEM / F-12) to a concentration of 5 × 10 ⁵ cells / mL. did. Dispensed 200 μL each in a 96-well culture plate (Nunc) was cultured in a carbon dioxide incubator (37 ° C., 7% CO ₂ ) for 24 hours. The fraction obtained by HPLC was concentrated with a centrifugal evaporator, dissolved in an appropriate amount of water, 2 μL thereof was administered to GT1-7 cells, and then an aqueous zinc solution was administered to a concentration of 30 μM. Cell viability was measured by WST-1 method 24 hours after zinc administration (n = 6). As a reagent, Cell Counting Kit was used.

  Centrifugal supernatant (0.3 mL) of urume sardine broth (IWA) was separated with a preparative ODS column, and fractions were obtained every 30 seconds. As a result of investigating the activity of inhibiting the nerve cell death of zinc for each of the concentrated fractions, the inhibitory activity was observed in the dotted line part (Fr1-6) in FIG. These fractions (IWA-C18_1-6) were collected several times and concentrated by lyophilization. Next, this fraction was separated by Amido-80, which is a normal phase column. Fractions were obtained for each peak, and the activity of inhibiting zinc neuronal cell death was examined for each concentrated fraction. Inhibitory activity was observed in the dotted line part (Fr9) in FIG. This fraction (IWA-AM_9) was collected several times and concentrated by lyophilization.

Example 4
The isolated active ingredient, IWA-AM_9, was identified by instrumental analysis.

IWA-AM — 9 was dissolved in a small amount of 30% acetonitrile and 0.05% trifluoroacetic acid solution, and mass spectrometry was performed. The mass spectrometer was analyzed in positive mode using LCQ Advance (Thermo Scientific) equipped with an ESI ionizer.
The results are shown in FIG. As a result of analysis between mass-to-charge ratios of 100 to 1000, a strong signal was obtained at m / z 156.1. Among the low-molecular components often contained in boiled juice and the like, histidine having the following chemical structure was a candidate as a structure having a molecular weight of 155.1.

  Since histidine, which is one of the amino acids, was considered as a candidate for the active ingredient, amino acids contained in IWA-AM_9 were then analyzed by amino acid analysis. The amino acid analysis was carried out using 7-Fluoro-7-nitrobenzofurazane (NBD-F) and the amino acid derived into the phosphor. IWA-AM — 9 was dissolved in a small amount of water and reacted with NBD-F at 60 ° C. for 1 minute under weak alkalinity. 20 μL of the reaction solution was detected at an excitation wavelength of 470 nm and a fluorescence wavelength of 530 nm by a gradient HPLC equipped with an ODS column (Shiseido Capsule Pack UG-120 C18, φ4.6 × 150 mm). A solution prepared by mixing acetonitrile and 75 mM phosphoric acid in a ratio of 16 and 84 was used as an eluent A, and a solution prepared by mixing acetonitrile, methanol, and 50 mM potassium phosphate in a ratio of 21, 39, and 40 was used as an eluent B for 0 to 15 minutes. : B 0%, 15-20 minutes: B 0-80%, 20-30 minutes: B 80-100%, 30-35 minutes: B 100%.

  FIG. 5 shows the results of amino acid analysis using Urumei boiled broth IWA-AM_9 as a sample. In addition, in the following including FIG. 5, amino acids etc. may be described in the following abbreviations in parentheses.

  That is, histidine (His), histamine (Hst), glycine (Gly), valine (Val), isoleucine (Ile), leucine (Leu), serine (Ser), methionine (Met), phenylalanine (Phe), aspartic acid ( Asp), arginine (Arg), alanine (Ala), proline (Pro), glutamic acid (Glu), glutamine (Gln), taurine (Tau), asparagine (Asn), β-alanine (β-Ala).

  As shown in FIG. 5, a large peak is observed at about 2.8 minutes, and it can be seen that it is histidine by comparison with a standard product. Since the peak appearing at about 10.6 minutes is a peak derived from the NBD-F reagent, it became clear that this active ingredient contains only histidine as an amino acid.

  By the way, most of the amino acids contained in the living body are in L form, but urume sardine broth is extracted by applying heat, so there is a possibility that not only L-histidine but also D-histidine, which is an enantiomer thereof, can be produced. D / L analysis of amino acids was performed using orthophthalaldehyde (OPA) and BOC-L-cysteine. IWA-AM_9 was dissolved in a small amount of water, added to a solution containing OPA and BOC-L-cysteine, and allowed to react at room temperature for 4 minutes. 20 μL of the reaction solution was detected at an excitation wavelength of 344 nm and a fluorescence wavelength of 443 nm by HPLC equipped with an ODS column (Nova Pak C18, φ3.9 × 250 mm). As an eluent, a mixture of acetonitrile and 0.1 M acetate buffer (pH 6.0) in a ratio of 11 to 89 was used.

  As shown in FIG. 6 (a), L-histidine (L-His) and its enantiomer D-histidine (D-His) are 13.4 minutes and 12. Appeared at 5 minutes. When IWA-AM_9 was used as a sample, as shown in FIG. 6B, a peak was observed only at 13.4 minutes, and no peak was observed at 12.5 minutes. This result shows that IWA-AM_9 contained only L-form L-histidine as histidine.

(Example 5)
Using histidine alone, the concentration dependence of zinc neuron death inhibitory activity was examined. L-histidine hydrochloride was dissolved in water. Immortalized hypothalamic neurons (GT1-7 cells) are cultured by a conventional method, dispersed with trypsin enzyme, and then added to a serum-free medium (DMEM / F-12) to a concentration of 5 × 10 ⁵ cells / mL. Thereafter, 200 μL each of which was dispensed into a 96-well culture plate (Nunc) was cultured in a carbon dioxide incubator (37 ° C., 7% CO ₂ ) for 24 hours. The prepared histidine solution was administered to GT1-7 cells so that the final concentration was 30 μM to 500 μM, and then 30 μM zinc aqueous solution was administered. Cell viability was measured by WST-1 method 24 hours after zinc administration (n = 6). As a reagent, Cell Counting Kit was used. As is clear from FIG. 7, cell death was suppressed depending on the histidine concentration, and addition of 300 μM histidine almost completely suppressed neuronal cell death due to zinc.

  As described later in Example 6, it has been clarified that 25 mM histidine is contained in urume sardine broth (100 mg / mL). Urumei boiled broth was serially diluted to 1/5 and 1/10, and the nerve cell death inhibitory activity of zinc was examined. When these diluted samples are added to the cell culture, the final concentration of histidine is 250 μM for the stock solution, 50 μM for the 1/5 dilution, and 25 μM for the 1/10 dilution. When this is put on the inhibitory activity graph of the histidine single product (FIG. 7), it is plotted at a point indicated by a black circle, which is in good agreement with the concentration dependence of the histidine single product. This result shows that the inhibitory active ingredient in urume sardine broth is only histidine.

(Example 6)
FIG. 8 shows the results of amino acid analysis of urume sardines. The amino acid analysis in FIG. 8 revealed that only a large amount of histidine was contained as an amino acid. It was revealed that 250 mg of histidine was contained in the 1 mg / mL sample, and that 38 mg of histidine was contained in 1 g of dried Urume sardine. 3.8% of the total weight is histidine, and it can be said that the dried urine simmered juice is useful in isolating histidine. In addition, it was found that the peak appearing at 4.5 minutes was taurine by comparison with the standard product, but taurine does not exhibit neuronal cell death inhibitory activity due to zinc.

(Example 7)
The histidine content of other seafood extracts that showed inhibitory activity was measured by amino acid analysis. The results are shown in Table 3.

The cell viability in Table 3 represents the viability when a sample and 30 μM zinc were added. The cell viability when only zinc was added was 10%. ND in the table indicates that measurement is not performed.
Concentration of the histidine contained was 28 mM, which was the largest for horse mackerel, and 2 mM, which was the smallest for sea squid. The dose for cell culture is 5% (10 μL vs. 200 μL). The final concentration when these samples were added is shown. It became 1400 μM for horse mackerel and 100 μM for sea squid. As shown in FIG. 7, histidine has an activity of suppressing neuronal cell death caused by zinc at a final concentration of 100 μM. Therefore, these samples showing the activity except for the sea urchin which has not been measured are contained. It was concluded that histidine showed inhibitory activity.

(Example 8)
Amino acid and taurine were examined for the inhibitory activity of zinc on neuronal cell death. All L amino acids were used except for glycine and histidine. Each single product was dissolved in water and added to the cells to a final concentration of 1 mM. Immortalized hypothalamic neurons (GT1-7 cells) are cultured by a conventional method, dispersed with trypsin enzyme, and then added to a serum-free medium (DMEM / F-12) to a concentration of 5 × 10 ⁵ cells / mL. Thereafter, 200 μL each of which was dispensed into a 96-well culture plate (Nunc) was cultured in a carbon dioxide incubator (37 ° C., 7% CO ₂ ) for 24 hours. Each prepared solution was administered to GT1-7 cells to a final concentration of 1 mM, and then 30 μM of an aqueous zinc solution was administered. Cell viability was measured by WST-1 method 24 hours after zinc administration (n = 6). As a reagent, Cell Counting Kit was used.

  As shown in FIG. 9, L-histidine and D-histidine showed inhibitory activity. No other amino acid (except cysteine) showed inhibitory activity. Also, no activity was seen in taurine. It was revealed that the inhibitory activity is a phenomenon specific to histidine among amino acids. Moreover, as shown in FIG. 10, the concentration dependence of the inhibitory activity of histidine was almost the same between L-form and D-form.

Example 9
The inhibitory activity of histidine on zinc neuronal cell death was examined in rat primary cultured neurons.
The cerebral cortex was excised from a rat fetus at 18 days of gestation and dispersed with papain enzyme, and then Dulbecco's MEM (DMEM) medium was added to a concentration of 2 × 10 ⁵ cells / mL. Subsequently, 200 μL each dispensed in a 96-well culture plate (manufactured by Nunc) was cultured in a carbon dioxide incubator (37 ° C., 7% CO ₂ ). After 1 week of culture, the medium was replaced with a serum-free medium (DMEM) and cultured for about 1 week. To the prepared rat hippocampal primary cultured neurons, 1% of L-histidine prepared to 100 mM was added and administered to a final concentration of 1 mM, followed by administration of 150 μM zinc aqueous solution. Cell viability was measured by WST-1 method 24 hours after zinc administration (n = 6). As a reagent, Cell Counting Kit was used.

  Similarly to the cell line GT1-7, as shown in FIG. 11, it was confirmed that histidine suppresses neuronal cell death caused by zinc even in primary cultured cells taken out from the rat brain. In addition, histidine having a final concentration of 1 mM almost completely suppressed neuronal cell death caused by zinc.

(Example 10)
Regarding analogs of histidine, constituent compounds, and histidine-containing compounds, the concentration at which zinc inhibits neuronal cell death was examined.
In addition to L-histidine and D-histidine, L-histidine amide, 1-methyl-L-histidine and acetyl-L-histidine were examined as analogs thereof. In addition, imidazole, which is a histidine side chain structure, and histamine, which is well known as a degradation product of histidine, were also examined. Furthermore, L-histidyl-L-alanine, L-alanyl-L-histidine, and β-alanyl-L-histidine (carnosine) were also examined as compounds containing histidine. Each single product was dissolved in water and added to the cultured cells so that the final concentration was 10 mM to 0.05 mM, and the inhibitory activity was examined. Immortalized hypothalamic neurons (GT1-7 cells) are cultured by a conventional method, dispersed with trypsin enzyme, and then added to a serum-free medium (DMEM / F-12) to a concentration of 5 × 10 ⁵ cells / mL. Thereafter, 200 μL each of which was dispensed into a 96-well culture plate (Nunc) was cultured in a carbon dioxide incubator (37 ° C., 7% CO ₂ ) for 24 hours. After the prepared solution was administered to GT1-7 cells, a zinc aqueous solution 30 μM was administered. Cell viability was measured by WST-1 method 24 hours after zinc administration (n = 6). As a reagent, Cell Counting Kit was used.

  For the compounds examined in Example 10, Table 4 summarizes the concentrations required to inhibit zinc neuronal cell death by 50%.

To summarize the results, the compounds examined in Example 10 can be classified into the following three groups.
The first group includes the following compounds having the same inhibitory activity intensity as histidine.

Ｌ−ヒスチジン

L-histidine

Ｄ−ヒスチジン

D-histidine

Ｌ−ヒスチジンアミド

L-histidine amide

Ｌ−ヒスチジル−Ｌ−アラニン

L-histidyl-L-alanine

  The second group includes the following compounds having the same inhibitory activity intensity as carnosine.

１−メチル−Ｌ−ヒスチジン

1-methyl-L-histidine

Ｌ−アラニル−Ｌ−ヒスチジン

L-alanyl-L-histidine

βアラニル−Ｌ−ヒスチジン（カルノシン）

β-alanyl-L-histidine (carnosine)

  As the third group, the following compounds having no inhibitory effect are listed.

イミダゾール

Imidazole

ヒスタミン

histamine

アセチル−Ｌ−ヒスチジン

Acetyl-L-histidine

  Comparing the above three groups, the amino group at the N-terminus and the amino group at the 1-position of the imidazole group are important for the activity of suppressing neuronal cell death caused by zinc, and either of them is blocked by methylation or the like It was revealed that the intensity of the inhibitory activity was greatly reduced. From these results, it has been clarified that histidine, its enantiomer, and / or a compound having histidine at the N-terminus strongly suppresses neuronal cell death of zinc.

Example 11
Enzymatic activity for decomposing D-amino acids is limited, and it is considered less susceptible to enzymatic degradation than L-amino acids. It was examined by HPLC whether D-histidine was present in the cell culture solution in a high concentration state for a long time compared to L-histidine.

  Table 5 shows the results of adding L-histidine and D-histidine to the cultured cells to a final concentration of 1 mM and evaluating how much histidine remains after 12 hours (residual ratio).

Immortalized hypothalamic neurons (GT1-7 cells) are cultured by a conventional method, dispersed with trypsin enzyme, and then added to a serum-free medium (DMEM / F-12) to a concentration of 5 × 10 ⁵ cells / mL. Then, 200 μL each dispensed in a 96-well culture plate (manufactured by Nunc) was cultured in a carbon dioxide incubator (37 ° C., 7% CO ₂ ) for 24 hours. The amount of histidine in the culture broth was quantified by amino acid analysis HPLC.
It was revealed that D-histidine showed less decrease in the initial stage of culture (0-12 hours) than L-histidine.

  For the prevention or improvement of cerebrovascular dementia caused by suppression of neuronal cell death by zinc released excessively in the brain after cerebral ischemia, the therapeutic agent for cerebrovascular dementia of the present invention, food and drink containing the therapeutic agent And supplements containing therapeutic agents are available. Compared to pyruvic acid, citric acid and carnosine, which have been known to have inhibitory activity against cerebrovascular dementia, histidine in the present invention exhibits inhibitory activity at a lower concentration. Moreover, histidine is a biological material and is widely used safely. Therefore, the present invention not only enables safe and long-term prevention and improvement of cerebrovascular dementia, which often requires long-term intake and medication, but also broadens the way of home treatment. Is also open.

It is a graph which shows the activity which suppresses the neuronal cell death of zinc by various seafood extracts. It is a figure which shows the progress of an active ingredient isolation | separation from Urume sardine soup with a reverse phase column. It is a figure which shows isolation of an active ingredient from Urume sardine soup by a normal phase column. It is a mass spectrometry figure which shows the identification of the active ingredient from Urume sardine soup. It is an amino acid analysis figure which shows the identification of the active component from Urume sardine soup. It is a figure which shows an amino acid D / L analysis, (a) is a standard product, (b) is a result of Urume sardine soup. It is a graph which shows the density | concentration dependence of the zinc nerve cell death inhibitory activity of histidine and an urine sardine broth. It is an amino acid analysis figure of Urume sardine broth. It is a graph which shows the zinc nerve cell death inhibitory activity of various amino acids. It is a graph which shows the density | concentration dependence of the zinc nerve cell death inhibitory activity of L-histidine and its enantiomer. It is a graph which shows the zinc nerve cell death inhibitory activity in the rat primary cultured neuron of histidine.

Claims (5)

  A therapeutic agent for cerebrovascular dementia, comprising histidine and / or a compound having histidine at the N-terminus as an active ingredient.   The therapeutic agent for cerebrovascular dementia according to claim 1, wherein the histidine is D-form.   The therapeutic agent for cerebrovascular dementia according to claim 1 or 2, wherein the histidine and the compound having the histidine at the N-terminus are derived from seafood.   A food or drink containing the therapeutic agent for cerebrovascular dementia according to any one of claims 1 to 3.   Supplementary food containing the therapeutic agent for cerebrovascular dementia according to any one of claims 1 to 3.

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